Terpenoids widely exist in higher plants, fungi, microorganisms, insects, and marine organisms in nature. More than 50 thousands of terpenoids have been discovered by now, most of which are biologically active ingredients in medicaments and health care products, for example, monoterpenes (C1O): Menthol, Linalool; sesquiterpenes (C15): Artemissinin; diterpenes (C20): Taxol, Tanshinone 11A; triterpenes (C30): Ginsenosides, Notoginsenosides, Glycyrrhizine; tetraterpenes (C40): Lycopene, β-carotene, Zeaxanthin, Cathaxanthin, and Astaxanthin, Carotenoides etc.; polyterpenes: Coenzyme Q10; terpenoid alkaloid: Dendrobine, Gentianine, Aconitine, Reserpine, and the like.
In view of that terpenoids are promising in a wide range of uses and have a great market demand, the methods for effectively producing terpenoids are always hot spots of present research. Currently, there are mainly three methods for producing terpenoids: chemical synthesis, plant extraction, and microorganism fermentation. Chemical synthesis has a complex process, high energy consumption, and severe pollution; on the other hand, terpenoids are often at a low amount in plants, and extraction from plant may cause serious damage to wild plant resource; in contrast, microorganism fermentation may be greatly advantageous due to no restriction from raw materials and being a friendly and clean production process.
There are two types of microorganism fermentation, and one of them uses wild strains. However, only a few of wild-type microorganism strains can produce terpenoids at present, and have relatively low productability of fermentation. Although traditional mutagenesis breeding may improve strain fermentation ability to some extent, it is more random, and less effective, and results in unstable mutant strain. The other one is fermentation using constructed recombinant strains. As continuous development in metabolic engineering and synthetic biological technology recently, more types of terpenoids are synthesized with recombinant strains in higher yield. The most commonly used recombinant strain is Escherichia coli (E. coli). In many studies, E. coli is selected as a starting strain for engineering because its genome sequence has been fully disclosed, it is clear in terms of genetic background and metabolic pathway, and it has many advantages such as simple demand for medium and fast growth (Ajikumar et al., 2008; Das et al., 2007; Keasling, 2008; Lee et al., 2002).
β-carotene and lycopene are two typical types of terpenoids. β-carotene has effects of antioxidation, detoxification, anticancer, preventing cardiovascular diseases, preventing and treating cataract, protecting liver, and the like. Moreover, β-carotene is also called “source of Vitamin A”, which is an important physiological function active substance in human, and is capable of treating epithelial cell keratinization, ophthalmoxerosis, nyctalopia, etc., caused by lack of Vitamin A (Lee et al., 2002; Das et al., 2007). Lycopene is the main constituent of red pigments of tomato, and is an excellent antioxidant. Lycopene is capable of preventing human tissues and organs from damage by a metabolic product of “free radical”, and may be used in natural heath foods or drugs. Lycopene-containing health foods are capable of preventing age-related deterioration of eyesight, resisting aging, and preventing cardiovascular diseases, and also exert some inhibition to each of digestive tract, cervical, breast, skin, bladder cancers and the like. Lycopene is nontoxic and harmless, and may be added to food such as ice cream, fruit syrup, hard candy, bread, cookie, cake, etc., like β-carotene, so as to improve the nutritional value thereof. It is superior to Vitamin E and β-carotene in health care effect.
Many natural microorganism, e.g., Blakeslea trispora, Pantoea agglomerans, Phaffia rhodozyma, etc., can synthesize β-carotene and lycopene (Mehta et al., 2003). DMAPP is successively react with 2 molecules of IPP, under the action of farnesyl diphosphate (FPP) synthase, to form FPP; FPP and IPP react, under the action of geranyl-geranyl diphosphate (GGPP) synthase, to form GGPP. Phytoene is synthesized head-to-head from 2 molecules of GGPP under the action of phytoene synthase (CrtB); lycopene is formed from phytoene under the action of phytoene desaturase (CrtI); and β-carotene is formed from lycopene under the action of β-lycopene cyclase (crtY).
Isopentenyl pyrophosphate (IPP) and dimethylallyl pyrophosphate (DMAPP) are precursor compounds of all terpenoid compounds. There are two synthetic pathways known so far (Lee et al., 2002). One is Mevalonic Acid Pathway (MVA pathway), mainly found on cytosol or endoplasmic reticulum in fungi and plants. Through this pathway, three molecules of acetyl coenzyme A are condensed to form 3-hydroxy-3-methyl-glutaryl coenzyme A (HMG-COA), and subjected to a two-step kinase reaction to form mevalonate pyrophosphate (MVA), and finally subjected to multi-step phosphorylation and decarboxylation reactions to produce IPP. The other one is MEP pathway, mainly found in bacteria, green alga and plant plasmids. This pathway is started with a starting material of 3-phosphoglyceraldehyde and pyruvic acid, and catalyzed with 1-deoxy-D-xylulose-5-phosphate synthase (Dxs) and 1-deoxy-D-xylulose-5-phosphate reductoisomerase (Dxr) to form 2-C-methyl-D-erythritol-4-phosphate (MEP). MEP is then formed into 2-C-methyl-D-erythritol-2,4-cyclodiphosphate (MEC), through three successive reactions catalyzed by 4-diphosphocytidy 1-2C-methyl-D-erythritol synthase (IspD), 4-diphosphocytidyl-2-C-methyl erythritol kinase (IspE) and 2C-methyl-D-erythritol 2,4-cyclodiphosphate synthase (IspF). MEC is then is formed into (E)-4-hydroxy-3-methyl-2-butenyl-diphosphate (HMBPP) under the action of (E)-4-hydroxy-2-methyl-2-butenyl4-diphosphate synthase (IspG). Next, HMBPP is catalyzed with HMBPP reductase (1-hydroxy-2-methyl-2-(E)-butenyl-4-diphosphate reductase, IspH) to form a mixture of IPP and DMAPP of 5:1. IPP is catalyzed with isopentenyl diphosphate isomerase (Idi) to be isomerized into dimethyl allyl pyrophosphate, DMAPP. IPP and DMAPP are basic C5 units of terpenoids, based on which various terpenoids may be synthesized.
In order for production of a terpenoid from E. coli, it is first necessary to introduce genes for synthesizing the terpenoid into E. coli. However, due to poor ability of E. coli to synthesize IPP and DMAPP, it always leads to a low terpenoid production. For improving the ability of synthesizing terpenoid by recombinant E. coli, it is required to enhance the ability to synthesize IPP and DMAPP thereof. Currently, there are mainly two solutions for enhancing the ability of recombinant E. coli to synthesize IPP and DMAPP: one being to introduce a foreign MVA pathway (e.g., the MVA pathway from Saccharomyces cerevisiae) (Martin et al., 2003; Yoon et al., 2007; Yoon et al., 2009); the other one being to improve the efficiency of E. coli own MEP pathway by increasing the expression strength of a key gene in MEP pathway and increasing supplies of precursor compounds of MEP pathway, 3-phosphoglyceraldehyde and pyruvic acid (Ajikumar et al., 2010; Alper et al., 2005a; Alper et al., 2005b; Choi et al., 2010; Jin and Stephanopoulos, 2007; Yuan et al., 2006).
In increasing the expression strength of a key gene in MEP pathway, previous attempts all focused on use of strong promoters (such as T5 promoter) (Yuan et al., 2006). Studies show that overexpression of 1-deoxy-D-xylulose-5-phosphate synthase gene (dxs), 4-diphosphocytidyl-2C-methyl-D-erythritol synthase gene (ispD) and 4-diphosphocytidyl-2-C-methyl-D-erythritol kinase gene (ispF), 2-C-methyl-D-erythritol-2,4-cyclopyrophosphate synthase gene (ispE), isopentenyl diphosphate isomerase gene (idi) in MEP pathway can improve the ability of E. coli in production of β-carotene. However, increase in the expression intensities of 1-deoxy-D-xylulose-5-phosphate reductoisomerase gene (dxr), 1-hydroxy-2-methyl-2-(E)-butenyl-4-diphosphate synthase gene (ispG), 1-hydroxy-2-methyl-2-(E)-butenyl-4-diphosphate reductase gene (ispH) in MEP pathway reduces the ability of recombinant E. coli in production of β-carotene (Yuan et al., 2006).
In another aspect, nicotinamide adenine dinucleotide phosphate (NADPH) and adenosine triphosphate (ATP) are key cofactors in MEP pathway. Insufficient supply of the cofactors will result in low efficiency in MEP pathway.